lncRNAs as Organizers of and Bridges Between Proteins and DNA

lncRNA 作为蛋白质和 DNA 的组织者和桥梁

• 批准号: 9356528
• 负责人: Robert T Batey
• 金额: $ 37.17万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-22 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9356528
• 关键词: Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biology; C-terminal; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromosomes; Code; Complex; DNA; DNA Binding; DNA Binding Domain; DNA-Binding Proteins; Data; Development; Diagnostic; Disease; Elements; Enhancers; Estrogen Receptor alpha; Exhibits; Female; Gene Dosage; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Glucocorticoid Receptor; Goals; Gold; Health; Heterogeneous-Nuclear Ribonucleoprotein U; High Mobility Group Proteins; Homeostasis; Human; Human Genome Project; In Vitro; Knock-out; Lamins; Large-Scale Sequencing; Lead; Link; Location; Malignant Neoplasms; Measures; Mediating; Metabolism; Modeling; Molecular; Mus; Mutation; N-terminal; Nature; Noise; Nuclear Envelope; Nuclear Hormone Receptors; Nucleotides; Play; Process; Proteins; RNA; RNA Binding; RNA Sequences; RNA-Protein Interaction; Recruitment Activity; Regulator Genes; Research; Resolution; Role; Site; Specificity; Starvation; Structure; Testing; Therapeutic; Tissues; Transcriptional Regulation; Translating; Untranslated RNA; Work; X Chromosome; X Inactivation; X-Ray Crystallography; base; ds-DNA; frontier; genome-wide; insight; intermolecular interaction; lipid biosynthesis; malignant breast neoplasm; pluripotency; programs; promoter; receptor binding; response; transcription factor; transcriptomics

Project Summary One of the unexpected developments in the last decade emerged from large scale sequencing efforts - the discovery of widespread genome-wide transcription. While the human genome project revealed an unexpectedly small fraction of the genome dedicated to protein-coding genes, the ENCODE and related projects revealed that at least 70% of the genome is actively transcribed. This led to the discovery of a new class of RNAs known as long non-coding RNAs or lncRNAs. Rapidly accumulating evidence strongly suggests that these lncRNAs have important autonomous activities as RNAs. The emerging functions are predominantly in development, differentiation and pluripotency, processes with critical links to human health. Thus, establishing an understanding of the mechanism of action of lncRNAs is a high priority frontier in biology. Towards the long term-goal of producing a molecular understanding of lncRNA function, we are using a set of biochemical and structural approaches to elucidate how lncRNAs organize aspects of the nucleus to regulate and coordinate chromatin expression. In the first Aim of our proposed research program, the molecular basis of the interaction between hnRNP U and the Xist and Firre RNAs will be investigated. These lncRNAs are essential for X-chromosome inactivation (Xist) and adipogenesis (Firre), using mechanisms requiring their direct interaction with hnRNP U that localizes the lncRNA to the correct chromosome or loci. Selective binding of the RNA-binding RGG domain of hnRNP U will be explored using a combination of "bottom-up" and "top-down" biochemical approaches and structural approaches to understand the molecular details of protein-RNA recognition, focusing on the critical but poorly understood RGG domain. The results of these studies will illuminate a number of RNA-protein interactions mediated by RGG domains that are central to human RNA metabolism. The second Aim focuses on the decoy/guide model for lncRNA function by investigating the interactions of two key transcription factors, glucocorticoid receptor (GR) and Sox2, with the lncRNAs that are proposed to modulate the specificity and regulatory activity of these proteins. According to this model, pervasive transcription at promoters and enhancers can either titrate away a transcription factor from its dsDNA-binding site or, conversely, help to recruit and localize a transcription factor to a target. To reveal the sequences and/or structures of lncRNAs that these dsDNA-binding proteins can recognize, an in vitro selection based approach will be used and the results correlated with transcriptomic studies. In parallel, traditional biochemical and structural approaches will be used to understand the molecular details of these protein-RNA interactions with known lncRNA targets. These studies will yield direct insights into how key transcription factors that have classically been regarded as solely dsDNA-binding factors also interact with RNA as a critical part of their gene regulatory mechanism.

项目概要 过去十年中出现的意想不到的发展之一是大规模测序工作—— 发现广泛的全基因组转录。虽然人类基因组计划揭示了 出人意料的是，基因组中一小部分专门用于蛋白质编码基因、ENCODE 和相关基因 项目显示至少 70% 的基因组处于活跃转录状态。这导致了一种新的发现 一类 RNA 称为长非编码 RNA 或 lncRNA。迅速积累的证据强烈表明 这些lncRNA与RNA一样具有重要的自主活性。新兴功能主要是 发育、分化和多能性，这些过程与人类健康有着重要的联系。因此， 建立对 lncRNA 作用机制的理解是生物学的一个高度优先的前沿领域。 为了实现对 lncRNA 功能的分子理解的长期目标，我们正在使用一组 阐明lncRNA如何组织细胞核各个方面以进行调节的生化和结构方法 并协调染色质表达。 在我们提出的研究计划的第一个目标中，hnRNP U 之间相互作用的分子基础 Xist 和 Firre RNA 将被研究。这些 lncRNA 对于 X 染色体失活至关重要 (Xist) 和脂肪生成 (Firre)，使用需要它们与本地化的 hnRNP U 直接相互作用的机制 lncRNA 到正确的染色体或基因座。 hnRNP U 的 RNA 结合 RGG 结构域的选择性结合 将使用“自下而上”和“自上而下”的生化方法和结构相结合的方式进行探索 了解蛋白质-RNA 识别的分子细节的方法，重点关注关键但效果不佳的 了解RGG域。这些研究的结果将阐明许多 RNA-蛋白质相互作用 由 RGG 结构域介导，该结构域是人类 RNA 代谢的核心。 第二个目标通过研究两个因子的相互作用来关注 lncRNA 功能的诱饵/引导模型 关键转录因子、糖皮质激素受体 (GR) 和 Sox2，以及建议的 lncRNA 调节这些蛋白质的特异性和调节活性。根据这个模型，普遍 启动子和增强子处的转录可以从其 dsDNA 结合滴定转录因子 位点，或者相反，帮助招募转录因子并将其定位到目标。揭示序列和/或 这些 dsDNA 结合蛋白可以识别的 lncRNA 结构，一种基于体外选择的方法 将被使用并将结果与​​转录组研究相关。与此同时，传统的生化和 结构方法将用于了解这些蛋白质-RNA 相互作用的分子细节 已知的 lncRNA 靶标。这些研究将直接洞察关键转录因子如何发挥作用 传统上被认为是唯一的 dsDNA 结合因子，也作为其基因的关键部分与 RNA 相互作用 监管机制。